After treating LFs with 25 µg mL-1 GNP-HCIm and 10 µM Imatinib for 24 h, cells were lysed. Proteins (10 µg) were loaded onto SDS-PAGE and transferred onto a PVDF membrane using a Trans Blot turbo system (Bio-Rad). After 1 h incubation in 5% Bovine Serum Albumin (BSA) diluted in PBS and three washes with PBS containing 0.1% Tween 20 (TBST), the membrane was incubated overnight with anti-c-Abl (phospho Y412) antibody (ab4717, Abcam) at a 1:1000 dilution in 1% BSA. After three washes with TBST (10 mL), membranes were incubated with Rabbit anti-Mouse IgG H&L (HRP) (ab6728, Abcam) secondary antibody at a 1:2000 dilution in 1% BSA in TBST, for 1 h at room temperature. Clarity Western ECL (Bio-Rad) solution was used according to the provided protocol. The same procedure was applied to identify c-Abl by anti-c-Abl antibody (ab15130, Abcam) at a 1:100 dilution and β-actin (15G5A11/E2, Cat #MA1-140, Thermo Fisher Scientific) at a 1:5000 dilution. Also in this case we used as secondary antibody Rabbit anti-Mouse IgG H&L (HRP) (ab6728, Abcam). All immunoblots were acquired with the ImageQuant LAS 4000 analyzer (GE Healthcare).
Ab6728
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab6728 is a primary antibody product from Abcam. It is a mouse monoclonal antibody that recognizes the human CD4 protein. The antibody is suitable for use in various immunoassay applications, such as flow cytometry and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (75 mentions)
Ab8245, by Abcam (16 mentions)
Pvdf membrane, by Merck Group (16 mentions)
Ab8226, by Abcam (11 mentions)
Ripa buffer, by Beyotime (11 mentions)
Ab181602, by Abcam (9 mentions)
Ab205718, by Abcam (9 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Las 4000, by Cytiva (7 mentions)
Ripa lysis buffer, by Beyotime (7 mentions)
Ab32124, by Abcam (7 mentions)
Bca kit, by Beyotime (7 mentions)
Ab9485, by Abcam (7 mentions)
Ab8227, by Abcam (6 mentions)
Image lab software, by Bio-Rad (6 mentions)
Ab32503, by Abcam (6 mentions)
Polyvinylidene fluoride membrane, by Merck Group (5 mentions)
Ab6276, by Abcam (5 mentions)
Imagequant las 4000 analyzer, by GE Healthcare (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
212 protocols using ab6728
1
Quantification of Phosphorylated c-Abl in Cells
2
Quantification of BACH1 Protein Levels
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Protein knockdown was measured with Western blotting using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific), a ChemiDoc MP Imaging System (Bio-Rad), and ImageLab version 5.2.1 (Bio-Rad). BACH1 was detected using the F-9 antibody (Santa Cruz Biotechnology, sc-271211) at 1:300 dilution and rabbit anti-mouse-HRP (Abcam, ab6728) at 1:5000 dilution. For normalization, actin was detected using anti-β-actin (Abcam, ab8227) at 1:10,000 dilution and rabbit anti-mouse-HRP (Abcam, ab6728) at 1:10,000 dilution.
3
Western Blot Analysis of MAPK Signaling
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Cells were lysed in RIPA buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail (Roche, USA). The protein concentration was measured using a BCA kit, and 30 μg of protein per sample was used for polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were blocked with fat-free milk or 5% BSA for 1 h at room temperature and then incubated with various antibodies: ERK5 (#3372, Cell Signaling Technology, 1 : 1000), phosphorylated-ERK5 (#07-507, Millipore, 1 : 1000), MEF2C (#5030T, Cell Signaling Technology, 1 : 1000), phosphorylated-MEF2C (sc-377535, Santa Cruz, 1 : 200), ERK1/2 (#9102, Cell Signaling Technology, 1 : 1000), p-ERK1/2 (#9101, Cell Signaling Technology, 1 : 1000), and β-actin (ab8226, Abcam, 1 : 1000) overnight at 4°C. After the membranes were washed with TBST, they were incubated with HRP-linked secondary antibodies: goat anti-rabbit IgG (#7074, Cell Signaling Technology, 1 : 2000) or goat anti-mouse IgG (ab6728, Abcam, 1 : 2000) for 1 h at room temperature. The density of the protein bands was determined by ImageJ software. Densitometric values of the ERK5, MEF2C, and ERK1/2 bands were normalized to that of β-actin. Phosphorylated protein levels were normalized to the respective total protein levels.
4
Protein Lysate Preparation and Western Blot Analysis
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Protein lysates were generated using RIPA buffer with PMSF (P0013B, Beyotime, Shanghai, China) and protein concentrations were quantified by BCA Protein Assay (P0011/P0012, Beyotime). Lysates were loaded on SDS-PAGE gels for transfer to a PVDF membrane, followed by incubation with the primary antibodies (Table S3 ) and then with HRP-labeled rabbit anti-mouse IgG (ab6728, Abcam, Cambridge, UK; 1:2000-1:10000) or HRP-labeled goat anti-rabbit IgG (ab6721, Abcam, 1:5000); GAPDH (ab181602, Abcam,1:10000) served as the internal reference. Enhanced chemiluminescence (P0018M, Beyotime) was added, and an ImageQuantLAS4000C gel Imager (GE, USA) was used for development (Li et al. 2018 (link)).
5
Immunohistochemistry for Ki67 and GAPDH
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The samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sliced into thin sections (5 µm thickness). After being dewaxed and rehydrated, the sections were incubated with 3% H2O2 for 30 min to block the endogenous peroxidase (POD) activity. Following antigen retrieval (AR) by microwave (heating), 5% bovine serum albumin (BSA) was applied to block non-specific binding. The sections were then incubated with the primary antibodies at 4°C overnight. Anti-Ki67 (ab15580) and anti-GAPDH (ab181602) were obtained from Abcam and used at the following dilutions: anti-Ki67 (1:1,000) and anti-GAPDH (1:3,000). After being rinsed with PBS 3 times for 5 min each, the sections were treated with biotinylated secondary antibody (1:200; ab6728; Abcam) for 1 h, followed by incubation with streptavidin-horseradish peroxidase (HRP) at 37°C for 20 min. Diaminobenzidine (DAB) substrate was used as a color developing agent for the visualization of Ki67-positive cells.
6
Exosomal protein profiling by Western blot
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Exosomes or cells were lysed with radioimmunoprecipitation assay (RIPA; Thermo Fisher Scientific, Waltham, MA, USA) buffer containing a complete protease inhibitor tablet (Roche, Mannheim, Germany). The lysates were cleared by centrifugation at 14,000 × g for 20 min and the supernatant fraction protein concentrations detected using a Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Next, 30 μg of lysate was electrophoresed by 10% SDS-PAGE, electro-transferred onto immobilon polyvinylidene membranes (Sigma-Aldrich, St. Louis, MO, USA), blocked for 1 h, and incubated overnight at 4°C with the following primary antibodies: TSG101 (1:1,000, ab125011), CD63 (1:1,000, ab216130), HSP70 (1:1,000, ab2787), SOCS1 (1 μg/mL, ab62584), SOCS2 (1 μg/mL, ab3692), and SOCS3 (1 μg/mL, ab16030; Abcam, Cambridge, UK); JAK2 (1:1,000, #3230), pJAK2 (1:1,000, #4406), STAT3 (1:1,000, #30835), pSTAT3 (1:1,000, #9145), and GAPDH (1:1,000, #2118; Cell Signaling Technology [CST], Danvers, MA, USA); and HMBOX1 (1:500, PA5-21558; Thermo Fisher Scientific, Waltham, MA, USA). Horseradish peroxidase (HRP)-linked anti-mouse (1:2000, ab6728) and anti-rabbit (1:2000, ab6721; Abcam, Cambridge, UK) immunoglobulin G (IgG) were used as secondary antibodies.
7
Protein Expression Analysis by Western Blot
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The cells were lysed in 10% sodium dodecyl-sulfate (SDS) containing 1% protease inhibitor (BL612A; Biosharp, Hefei, China), and the protein concentration was quantified via Bradford protein assay. A total of 10 µg of protein lysates from each sample was separated in 10% SDS–polyacrylamide gel electrophoresis (PAGE) and then transferred onto polyvinylidene fluoride membranes (IPVH00010; MilliporeSigma, Burling, MA, USA). After blocking was completed with 5% skim milk, the membranes were incubated overnight in a 4 ℃ refrigerator with the primary antibodies against SLC16A1 (20139-1-AP; Proteintech, Wuhan, China) and GAPDH (KM9002; Sungene Biotech, Tianjin, China). After being washed, the membranes were probed with anti-rabbit immunoglobin G (IgG) secondary antibody (ab205718; Abcam, Cambridge, UK) or anti-mouse IgG secondary antibody (ab6728; Abcam).
8
Immunoblotting of C-terminal FLAG-tagged RutR
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For immunoblotting of C-terminally FLAG-tagged RutR protein, bacterial cells were sonicated in lysis buffer (100 mM K2HPO4/KH2PO4 pH 7.4, 150 mM NaCl, 1 mM EDTA, 20 mM nicotinamide, 2.5% Triton X-100, 5 mM AEBSF, 5 mM BA). Lysates were resolved by SDS-PAGE and analyzed by immunoblotting using an anti-FLAG-AB (Invitrogen, FG4R) primary antibody and a rabbit anti-mouse-HRP secondary AB (abcam, ab6728). Immunoblotting was conducted using a standard protocol. Detection was done by using enhanced chemiluminescence (Roth). Immunoblot analysis was done by measuring mean grey intensities using ImageJ software (http://rsbweb.nih.gov/ij ). For statistical analyzes, a two tailed sudent’s t-test was applied.
9
Bacterial Expression Vectors for Protein Modification
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For expression in bacterial cells pRSFDuet-1 (Merck/Sigma-Aldrich, Novagen) and a vector derived from pGEX-4T-1 (GE Healthcare) were used and modified as described above (Supplementary Data 4 ). Mutations were introduced by site-directed mutagenesis according to QuickChange protocol (Supplementary Table 8 ). For cloning, Phusion-DNA-polymerase, Taq-DNA-ligase, T5 exonuclease, and restriction enzymes were used (New England Biolabs). Anti-AcK-, anti-His6-, anti-FLAG-primary antibodies and suitable HRP-coupled secondary antibodies were purchased from Abcam, Cell Signaling Technologies and Invitrogen (rabbit anti-AcK-AB: abcam, ab21623/dilution: 1:1000 in 3-5% (w/v) milk; mouse anti-His6-AB: abcam, ab18184/dilution: 1:000 in 3-5% (w/v) milk; rabbit anti-FLAG-AB: Cell Signaling Technology, CST14793/dilution: 1:1000 in 3–5% (w/v) milk; mouse anti-FLAG-AB: Invitrogen, FG4R/dilution: 1:2000 in 3% (w/v) milk; goat anti-rabbit-HRP-AB: abcam, ab6721/dilution: 1:10000 in 3-5% milk; rabbit anti-mouse-HRP-AB: abcam, ab6728/dilution: 1:10000 in 1-5% (w/v) milk).
10
Western Blot Analysis of c-Myb and PKMYT1
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The cells were first lysed with a RIPA lysate and then the concentration of proteins was evaluated via a BCA protein assay kit (Thermo Fisher Scientific). Denaturation of protein by boiling and protein loading buffer. 10% SDS-PAGE was utilized to separate the proteins, which was then transferred to a polyvinylidene fluoride (PVDF, Millipore, Billerica, MA, USA) membranes. After incubated with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with the primary antibodies against c-Myb (1:100, Abcam, ab169111) and PKMYT1 (1:100, Abcam, ab200387) at 4 °C overnight. Then the membranes were incubated with secondary antibody (1:50,000, Abcam, ab6728 or ab6721) conjugated with horseradish peroxidase at room temperature for 1 h. Finally, the Enhanced Chemiluminescence Detection System (Thermo Fisher Scientific) was applied to detect the protein signals. GAPDH served as internal control.
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